I am setting up reverse transcriptase qPCR on total RNA to measure the Ct values in the cDNA. I am using GAPDH as the endogenous gene to measure. I have a relative standard curve in the plate as well using internal control RNA to use for quantification. After the RT-qPCR measures the Ct values, how do I convert the Ct values to ng amount in this assay?
For example, I have my control RNA measure from 100ng/ul down to 0.001ng/ul in 10fold dilutions to make the standard curve. My qPCR reaction is 10uL. i add 1uL of 100ng/ul RNA into the qPCR. How much cDNA is there in the reaction? If I didn't know the starting concentration of the RNA, how can I calculate from the cDNA Ct values how much RNA there is in the original sample?